Potassium (K+) channels are an exceptionally diverse group of ion channels that are similar in their ability to select K+ over other ions, but differ in their kinetic, voltage-dependent, pharmacological and single-channel behavior. Due their prevalence and diversity in the nervous system, the distribution of different classes of K+ channels plays a leading role in controlling neuronal activity. Different classes of K+ channels have been shown to contribute significantly to several important physiological functions including action potential repolarization, cardiac pacemaking, neuron bursting and learning and memory. This proposal will examine the temporal and spatial expression patterns of different K+ channel subtypes in the nervous system. Long- term objectives are to correlate differences in K+ channel expression to differences in excitability properties of individual neurons. The project focusses on the Shaker (Sh) gene complex in the fruit fly, Drosophila melanogaster. Molecular analysis indicates that Sh encodes a family of functionally distinct K+ channels that are expressed from differentially spliced transcripts. Specific aims are to examine developmental, cellular and subcellular distribution of different Sh gene products. Antibodies specific for the divergent amino and carboxyl domains of Sh proteins will be raised against synthetic peptides and full-length Sh proteins expressed from plasmid expression vectors. The antibodies will be used to investigate temporal and spatial distribution of different Sh proteins by immunocytochemistry and immunoblot analysis. In addition, expression patterns of Sh transcripts will be examined by tissue in situ hybridization using radiolabeled probes containing unique sequences from the divergent 5' and 3' domains of Sh mRNAs. From the coordinated set of experiments, we will determine where and when: a) the Sh gene is transcribed, b) the RNAs are processed, and c) the proteins are expressed. Temporal and spatial patterns of Sh expression will be examined in various mutant backgrounds in order to identify those genes (or gene products) that perturb the normal developmental and tissue- specific expression pathways.